1. Field of the Invention
The invention relates to a method and apparatus for analyzing the concentration of a substance in a sample liquid by the technique of competitive binding analysis, a currently important subdivision of which is radioimmunoassay. The technique involves the combining of the substance to be measured (ligand) with a specific binding agent. The ligand is often an antigen and the binding agent an antibody thereto. The extend of the reaction is measured by a label or tracer, usually radioactive, but tracer may also be, for example, flurorescent or an enzyme. The present innvention relates in particular to methods and apparatus permitting such analysis to be made more rapidly by machine with a minimum of labor and error.
2. Description of the Prior Art
Yalow and Berson (Nature 1 84,1648,1959) introduced a new analytical method for assaying the minute amounts of insulin found in the blood. An antibody to insulin was mixed with the sample plus a known amount of radioactive insulin. The total concentration of insulin exceeded the binding capacity of the antibody. At equilibrium, when all the antibody was bound to either radioactive insulin or nonradioactive insulin, the antibody bound insulin was separated from the free insulin by membrane electrophoresis and the radioactivity in each portion measured. By means of standards a relationship was established between the ratio of bound to unbound radioactivity and the amount of insulin in the sample. Because the general analytical principle of the method is so exquisitely sensitive and specific for biologically important molecules that are difficult to analyze by other means, the method has grown and diversified into an important clinical procedure.
Specific binding agents now include cell membrane receptors, tissue receptors, naturaly occurring specific binding agents such as the transins as well as the more common antibodies.
Ligands include elements, peptide hormones, steroid hormones, proteins, virus and tumor components. In addition to radioactive labeling we also find fluorescent and other optical labels and enzyme labeling. In those cases where the binding agent is the analyte, a constant amount of ligand is employed.
Separation methods include: differential migration of bound and unbound ligand such as gel filtration, chromatography, zone electrophoresis; isolation of unbound ligand by adsorbtion on coated charcoal, silica, talc; isolation of bound ligand by double antibody precipitation, salt precipitation, ethanol precipitation, dialysis. The Skeggs (U.S. Pat. No. 2,797,149) continuous dialysis and the Ferrari (U.S. Pat. No. 3,211,645) continuous filtration techniques have not been employed for this separation. Solid phase systems employing binding agent affixed to test tube wall, beads or column packing have become popular.
Because of the increase in the volume and applications of this technique to clinical medicine, recent attention in this area has centered an automatic and semiautomatic systems for performing these analyses. Many of these are adaptations of the continuous chemical analyzer of Skeggs (U.S. Pat. No. 2,797,149). These include the devices marketed by Technicon Corp. which use magnetic retention of iron coupled antibody. Also the Brooker (U.S. Pat. No. 4,022,577) method of dynamic measurement of total radioactivity followed by a second static measurement of one separated component. Also the Johnson (U.S. Pat. No. 3,896,217) method of separation with alternate adsorbtion and elution of one component on a column containing antibody fixed to the column making for efficient reutilization of antibody. A need exists for a technique which combines automation with simplicity, versatility and greater throughput rate for samples.